ASSIGNMENT

ON

APPLICATION OF MOLECULAR
MARKERS

SUBMITTED
BY

PUNESH
(2009BS36D)
(Ph.D. BIOCHEMISTRY)

CCS HARYANA AGRICULTURE UNIVERSITY

HISAR
 APPLICATION OF MOLECULAR MARKERS

Crop improvement and breeding have undergone a tremendous shift to feed the
growing population in Asian countries. Breeding a new variety by using conventional
breeding methods will take about eight and twelve years, although the release of an improved
variety cannot be guaranteed. Conventional breeding is both time consuming and dependent
on environmental conditions. Novel and innovative methods of biotechnology such as tissue
culture, transformation, clonal propagation and molecular markers, have contributed to the
development of high yielding, nutrient-rich modified or new varieties. Plant tissue culture
played an important role in an improvement of perennial crops. In this article, we have
discussed the application of molecular markers in the plant tissue culture, marker assisted
selection and breeding.

Morphological markers in plants.

Marker in lifesciences is defined as a trait or an allele or a DNA sequence or a
cytogenetic segment or a chromosome fragment or a protein or an enzyme used as an
experimental probe to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or
a gene. In general, different types of markers are used in lifesciences. Markers are broadly
classified into morphological markers and molecular markers. Morphological markers (or
also called phenotypic markers) are those distinguishable traits that are evident to human
naked eyes. These markers are selected based on the experience of the breeder to correlate a
phenotypic trait with a trait of interest. Morphological markers differ among species, genus
and varieties of plants and animals. It is the easiest and quickest way to identify or detect the
presence of a morphological marker with that of a trait for improvement. For example: Many
morphological markers, originating mainly from mutational studies, have been assigned to
specific barley chromosomes. Morphological markers are detected and well established in
different members of Poaceae (grass family) family such as different growth, yield and dis-
ease resistance traits are related to markers such as purple hull, phenol staining, glabrous leaf
and hull, purple node, awned panicle, lazy growth habit, brown pericarp and others.

Molecular markers in plants.

Molecular markers have wide application in different branches of lifesciences. In
genetics, molecular marker is defined as a fragment of DNA sequence that is associated to a
part of the genome carrying genes responsible for a trait. It is usually defined as an allele or
DNA sequence or chromosome fragment indicating the existence of a metabolism or
chemical or physical process. Sometimes this category of markers is referred to as biomarkers
or biosignatures or molecular signatures. In medicine, it could be a substance that is
introduced into an organism as a means to detect something (Ex: rubidium chloride is used as
a radioactive isotope to evaluate perfusion of heart In biology, it could be a substance native
to the organism whose detection indicates a particular disease state (Ex: presence of an
antibody might indicate an infection). Molecular markers are also sub-divided into
Biochemical markers (Eg: Isozymes, Allozymes), DNA based markers (Eg: RFLP, RAPD,
SSRs), Physiological markers, Biomarkers and Protein markers (also called biochemical
markers) depending on the biochemistry, physiology and origin of markers within the plant.
Molecular markers are phenotypically neutral. This is a significant advantage compared to
traditional phenotypic markers.
Isozyme is a molecular marker system based on the staining of proteins with identical
function, but different electrophoretic mobilities. The weakness of Isozyme markers is that
each of the proteins that are being scored might not be expressed in the same tissue and at the
same time in development. Therefore several samplings of the genetic population need to be
made. Molecular markers have very rapidly complemented the classical strategies. Molecular
markers include biochemical constituents (e.g. secondary metabolites in plants) and
macromolecules, viz. proteins and deoxyribonucleic acids (DNA). Analysis of secondary
metabolites is restricted to those plants that produce a suitable range of metabolites which
could be easily analyzed and which could be distinguished between varieties. These
metabolites, which are being used as markers should be neutral to environmental effects or
management practices. Hence, amongst the molecular markers used, DNA markers are more
suitable and ubiquitous to most of the living organisms.

The following properties are desirable for ideal DNA markers:

• Codominant inheritance (determination of homozygous and heterozygous states of diploid
organisms)
• Easy access (availability) and Easy exchange of data between laboratories
• Easy and fast assay
• Frequent occurrence in genome
• High reproducibility and Highly polymorphic nature
• Selective neutral behaviour (the DNA sequences of any organism are neutral to
environmental conditions or management practices)
It is extremely difficult to find a molecular marker which would meet all the above
criteria. Depending on the type of study to be undertaken, a marker system can be identified
that would fulfill atleast a few of the above characteristics. Various types of molecular
markers are utilized to evaluate DNA polymorphism and are generally classified as
“hybridization based markers” and “polymerase chain reaction (PCR) based markers”. In the
former, DNA profiles are visualized by hybridizing the restriction enzyme digested DNA, to
a labelled probe, which is a DNA fragment of known origin or sequence. PCR-based markers
involve in vitro amplification of particular DNA sequences or loci, with the help of
specifically or arbitrarily chosen oligonucleotide sequences (primers) and a thermostable
DNA polymerase enzyme.

The following are the examples of DNA based molecular markers:

• Restriction Fragment Length Polymorphisms (RFLPs)
• Random Amplified Polymorphic DNAs (RAPDs)
• Amplified Fragment Length Polymorphisms (AFLPs)
• Simple Sequence Repeats (SSRs)
• Sequence Tagged Microsatellite Sites (STMS)
• Sequence Tagged Sites (STS)
• Single Nucleotide Polymorphism (SNP)
 The detailed description of development of molecular markers is published by
different peer reviewed articles. The comparison of different methods of estimating genetic
diversity could define their usefulness in plant breeding and genetic improvement programs.
RFLPs have been used the most extensively. RFLP, SSR and AFLP markers are most
effective in detection of polymorphism in plants. RFLP markers have several advantages in
comparison with the RAPD and Isozyme markers, such as (1) they are codominant and
unaffected by the environment, (2) any source DNA could be used for the analysis and (3)
mapping markers linked to traits in a population that has not undergone specific stress effects.
The primary drawback of RAPD markers is that they are dominant and do not permit the
scoring of heterozygous individuals. AFLPs and SSRs are more popular than RFLP. RFLP
process is laborious. SNP is chosen when sufficient amount of sequence information is
available and analysis tool is available to assign gene function.

Application of markers in plant genetics and tissue culture.

In commercial industry, molecular markers are used in the confirmation of genetic
fidelity in micropropagated tree species, where life span is quite long and performance of
micropropagated plants could only be ascertained after their long juvenile stage in field
conditions. RFLPs (using nDNA and cpDNA probes) and RAPDs are also used for
characterization and identification of genetic resources of perennial crops and to solve
problems related to plant genetic diversity conservation. Scientists have used markers in
detection of epigenetic variations originated during storage and conservation, (i.e. detect
duplications, seed mixtures, inadvertent outcrossing and genetic drift). SSR markers are used
to determine the degree of relatedness among individuals or groups of accessions, to clarify
the genetic structure, or partitioning of variation among individuals, accessions, population
and species of rice. Markers are used to detect genes for segregation distortion in anther
culture derived plants.

Genetic diversity
Assessment of genetic diversity is important in plant breeding if there is to be
improvement by selection. For assessment of genetic diversity, molecular markers have been
generally superior to morphological, pedigree, heterosis, and biochemical data (isozymes and
chromatography). Genetic diversity commonly is measured by genetic distance (GD) or
genetic similarity (GS = 1 - GD), both of which imply that there are either differences or
similarities at the genetic level.
Published applications of molecular marker-based GD in plant breeding have been
limited primarily to maize, but results should apply to other species. Melchinger (1993)
reviewed the application of molecular marker-based GD for assigning maize inbred lines to
heterotic groups, determining the relation between inbred lines and hybrids, and predicting
hybrid performance. Data showed that GS calculated from molecular marker data faithfully
separated inbred lines into their heterotic groups. There also seems to be promise of assigning
inbreds of unknown pedigree to heterotic groups although a large number of markers (> 100)
and well-characterized reference populations may be needed to obtain an accurate assessment
of GS. Strong correlations (0.61 to 0.95) between Malecot's coancestry coefficient (f) and GS
for related (f > 0) genotypes indicated that pedigree data provide reliable estimates of GS.
Genetic similarity estimates based on molecular markers are expected to be superior to
estimates of f because of unreliable or incomplete pedigree data and because of the
assumptions required to calculate f. Molecular marker-based GD has some potential for
predicting hybrid performance of related lines, but in typical hybrid-breeding programs, in
which hybrids are produced from unrelated lines from different heterotic groups, molecular
marker-based GD has been of no value in predicting hybrid performance. These results
suggest the use of molecular marker-based GD for predicting hybrid performance in crops in
which either hybrids are being explored, such as wheat (Triticum aestivum [Vill., Host]
Mackey), and soybean (Glycine max (L.) Merr.), or hybrid breeding is being practiced, but
distinct heterotic groups have not been developed.
Molecular marker-based GD also has potential for assessing changes in genetic
diversity over time, protection of intellectual property rights, registration of germplasm in
countries having ratified the rules of the UPOV convention, and evaluation of new sources of
germplasm for their potential to increase genetic diversity.
Direct applications to plant breeding, however, have been limited so far to prediction
of hybrid performance. But accurate prediction of hybrid performance does not seem likely
unless gene action is primarily dominant or overdominant, complementary heterotic groups
are established, trait heritability is high, at least 30 to 50% of the markers are linked to QTL,
and no more than 20 to 30% of the markers are dispersed randomly. Molecular markers,
however, may be useful for early generation testing in hybrid-breeding programs. If
individual markers or marker intervals associated with combining ability can be identified
when a plant or progeny is crossed onto a given tester, then these markers could be used as a
first screen to identify the top 50% of the progenies for field evaluation. Although this
procedure would not decrease the time to cultivar development, it would decrease the amount
of material tested or permit the evaluation of a wider range of germplasm for the same
amount of field resources.
An equally important application of molecular marker-based GD may be in the
selection of parents to cross in a breeding program. This application deserves serious
attention because breeders currently rely primarily on pedigree and performance data for
choosing parents in breeding programs. Using molecular markers to select parents has the
potential to allow simultaneous maintenance of genetic diversity and performance. Dudley et
al. (1992) presented one application of molecular markers for choosing parents, and
additional research is needed in this area. Using molecular markers to choose parents likely
will require establishment of a relation between GD and genetic variation, and many of the
same conditions necessary for predicting hybrid performance may be required for choosing
parents. Using molecular markers as a diagnostic tool to survey new or exotic germplasm for
novel genetic diversity also may be possible. It is unlikely, however, that this use will be
possible with random genomic or cDNA clones because molecular marker-based genetic
diversity will not guarantee genetic diversity for the traits of interest. Screening with probes
of expressed genes with known function offers the greatest potential in this area.

Mapping QTL
The vast majority of molecular marker research in quantitative traits has been devoted
to mapping QTL. Mapping QTL is really a misnomer, because what is actually being done is
the mapping of chromosomal regions containing one or more putative QTL. With current
mapping technology, the existence of a single QTL between two flanking markers cannot be
resolved clearly.
Most studies reported to date have detected, localized, and estimated genetic effects in
the same experiment because of resource limitations. Genetic effects of mapped QTL regions
are over estimated by this procedure because of sampling errors (Lande and Thompson, 1990;
Lande, 1992). Furthermore, few researchers have followed up with the necessary experiments
to verify the effect of a chromosomal region on phenotype across mapping populations. Our
purpose in this section is not to review QTL mapping studies in detail, but rather to outline
the steps taken in QTL mapping experiments, to demonstrate the general results that have
been obtained, to outline some of the problems in translating these results into plant
improvement, and to show the types of previously unattainable information that these results
have contributed. The first step in QTL mapping studies is to detect QTL, while minimizing
the occurrence of false positives (Type I errors, that is, declaring an association between a
marker and QTL when in fact one does not exist). Two distinct methods are used to detect
QTL. The single marker approach, sometimes referred to as the one-way analysis of variance
(ANOVA), has been used extensively, especially with isozymes. The second approach,
interval mapping, detects QTL by using flanking markers. This approach is more complicated
analytically than the ANOVA approach and involves application of the maximum likelihood
method, which requires sophisticated computer software.
Lander and Botstein (1989) have developed formulae for calculating significance
levels appropriate for both methods when the genome size, number of chromosomes, number
of marker intervals, and the overall false positive rate desired are given. Several statistical
procedures have been developed for the application of both ANOVA and interval mapping.
When the same false positive rates are used, there are few reasons to suspect that the two
methods would detect substantially different QTL. Stuber et al. (1992) compared the two
methods and found that they identified basically the same QTL. Those researchers reported,
however, some advantages to using the interval mapping approach. Because of the increased
power associated with using flanking markers, the method gives the most likely location of
the QTL under the assumption of a single QTL in the interval, and the interval mapping
approach allows ambiguous or missing data.
Once QTL are detected, the next step is to estimate the genotypic effect of the QTL
and to localize the QTL to a precise genomic region. The interval mapping approach seems
superior to the ANOVA approach for both estimation of effects and localization of the QTL.
The success of both methods depends on the linkage between marker(s) and QTL, the
number and type of progeny evaluated, the heritability of the trait, and the magnitude of the
effects at QTL that one desires to detect. Several methods and genetic designs have been
suggested for detecting, estimating effects, and localizing QTL.

Manipulating traits controlled by a few major loci

The manipulation of traits controlled by a few major loci (loci that can be studied
using Mendelian genetics) may offer the greatest promise in the short term for application to
plant breeding. The primary applications of this technique will be for traits controlled by a
single gene (monogenic) or those controlled by at most two or three loci (oligogenic). The
most successful applications will be in those species with well developed molecular marker
maps. These applications will be immediately useful for "defensive breeding," that is, when a
desirable genotype is available but lacks resistance to important insects and diseases. Other
applications, not fitting into the category of defensive breeding may include seed
modifications controlled by a few genes, restorer genes for cytoplasmic male sterility,
dwarfing genes for shorter plant height, and maturity genes for adaptability.
The first requirement of using molecular markers in this context is to develop a
precise molecular marker linkage map and then to use these markers to map gene(s)
controlling the trait of interest. Many methods for mapping genes of interest are available,
including a variety of applications suitable to most species with polymorphic markers. The
final step is to use marker-facilitated selection to transfer the gene(s) to the genotypes
desired. Two methods are available, both of which begin with inbred lines, backcross
selection, and pedigree selection. If the cost of molecular-marker technology is ignored, the
primary factor affecting the design and the success of marker-facilitated selection is how
tightly linked a single marker is to the gene or how tightly bracketed the gene is by two
markers. The idea is to obtain a marker or a set of flanking markers linked tightly enough to
the gene so that a recombination event does not occur between the marker and the gene
during backcrossing or pedigree selection. Melchinger (1990) presented extensive theoretical
and numerical results for the backcross method concerning the optimal family size and the
number of plants per family that must be genotyped with molecular markers. The results are
complicated and will have to be assessed on a case by case basis. The economics of marker
assisted backcrossing will be a function of the cost of marker assays, the cost of direct
screening, and the value of accelerating the backcrossing program. Results regarding sample
sizes required for pedigree selection are unavailable, although marker-gene linkage is the
primary consideration.
There are many practical applications of molecular markers to traits controlled by few
loci. Most of the applications involve situations in which either screening for the trait is
difficult or scoring of the trait occurs late in plant development. These applications may
include pests for which natural inoculum is unreliable or artificial inoculation procedures are
undeveloped or unreliable. Examples include nematodes or Aspergillus, both of which have
broad host ranges and unreliable natural and artificial inoculation. Diseases in which
resistance is influenced strongly by the environment also would be good candidates for
marker facilitated selection. Unfortunately, the very situations favoring marker-facilitated
selection also make it difficult to map the resistance genes precisely. Marker-facilitated
selection has been advantageous for backcrossing recessive genes, when progeny tests are
needed after every backcross generation to identify heterozygotes or when resistance can be
determined only after flowering. Markers in these situations could greatly reduce workload
and backcrossing time. Other examples include pyramiding resistance genes, developing
multilines in which many race-specific resistance genes are involved that are sometimes
difficult to distinguish, and selecting for resistance to exotic or quarantined pathogens.
One of the primary advantages of marker-facilitated backcrossing has been in
increasing the speed of recovery of the recurrent parent genome. In addition to having a tight
marker-gene linkage, one or more neutral polymorphic markers will be required per
chromosome arm. The idea is to screen for plants having the resistance gene and to identify
those plants with the greatest proportion of markers homozygous for the recurrent parent. A
possible limitation of this procedure is that there may be an increase in the number of plants
needing to be assayed with markers. The procedure could be applied, however, to subsequent
backcross generations to ensure the recovery of unlinked segments of recurrent parental
genome.

Manipulating traits controlled by many loci

The molecular marker manipulation of traits controlled by many loci (from a plant
breeding perspective many is generally greater than five) is of great interest to plant breeders
and represents one of the fields greatest challenges. Plant breeders concentrate effort on
breeding for quantitative traits, and breeding for qualitative traits is generally a trivial, albeit
time consuming, process. The matter is further complicated because breeders usually evaluate
simultaneously in many populations -- four or more complexly inherited traits. Obviously,
any technology enhancing the breeders' ability to select for these traits would be adopted
rapidly.The molecular marker manipulation of qualitative traits is feasible precisely because
so much is known about the biology of these traits. The number of loci is known; there are no
questions about what a major locus is -- in fact, many of these loci have been cloned; the
gene action is known precisely; epistatic interactions, if any, are relatively easy to decipher;
and interactions with environment are easy to determine. In short, the amount that can be
known about qualitative traits is limited only by one's desire to know. As pointed out earlier,
for quantitative traits, the answers to these questions are based only upon averages over an
unknown number of loci. At the outset, the manipulation of quantitative traits by molecular
markers has a serious disadvantage. Even with these limitations, there still may be many
applications of marker-QTL associations.
But very little theoretical or applied research has been published in this area. The
most-cited application has been marker-assisted selection (MAS) although the context of
application often has been ignored. In plant breeding, there are two distinct but related
applications: germplasm enhancement (recurrent selection) and cultivar or hybrid
development. These two applications are separated because recurrent selection usually is
applied to random mating populations possibly at or near linkage equilibrium, whereas
cultivar or hybrid development typically begins with populations derived by crossing elite
inbred lines at or near maximum linkage disequilibrium . Clearly, two different approaches
are needed for these breeding schemes. Lande and Thompson (1990) and Lande (1992)
investigated the efficiency of MAS for both individual and mass selection in random-mating
populations. There are three approaches to applying MAS to plant breeding: (1) selection on
markers alone with no measurement of phenotype; (2) simultaneous selection on markers and
phenotype; and (3) two-stage selection, the first stage involving use of markers to select
among seedlings and the second involving phenotypic selection among surviving adults. On
individuals, the efficiency of MAS relative to that of phenotypic selection of the same
intensity is (p/h2)1/2, where p is the proportion of the additive genetic variance accounted for
by markers, and h2 is the heritability. Selection on markers alone will be more efficient than
phenotypic selection only when the proportion of the genetic variance explained by markers
exceeds the heritability of the trait. Therefore, selection on markers alone will be most useful
for traits with low heritabilities when large proportions of their variability have been
explained by markers. Lande and Thompson (1990) concluded that molecular marker loci can
be used to enhance the efficiency of artificial selection for quantitative traits. The potential
efficiency of MAS depends upon the heritability of the trait, the proportion of the genetic
variance explained by the markers, and the selection method. A major practical problem in
using MAS is that recombination will reduce linkage disequilibrium between the markers and
QTL, thus diminishing selection effectiveness. The successful application of MAS will
require very tight linkages between markers and QTL.

Understanding the genetic architecture of quantitative traits

Although there have been few direct applications of molecular markers in plant
breeding, published research has begun to provide information on the genetic architecture of
quantitative traits. Molecular marker-mapping data from several species now suggest that
genetic variation for quantitative traits is controlled by a few loci with large effects and many
loci with progressively smaller effects (Lande, 1992). Beavis et al. (1991) found 14 genomic
regions associated with plant height in four F2 maize populations, but few of these regions
were in common across populations. All 14 regions, however, were associated with known
qualitative genetic loci. Thus, circumstantial evidence supports Robertson's (1985) hypothesis
that QTL have alleles with a range of effects, alleles with large effects causing recognition of
the locus as qualitative, and alleles with small effects causing recognition of the locus as
quantitative. The results from QTL-mapping studies regarding gene action and epistasis are,
so far, difficult to interpret. Stuber et al. (1992) reported that, with one exception, all the QTL
mapped for grain yield in maize showed heterozygote superiority. Either overdominance or
pseudo-overdominance, therefore, likely was important in the mapping population. These
results cannot separate the two causes of heterozygote superiority, primarily because the
number of QTL residing in a marker interval cannot be resolved. Although not mentioned by
the authors, heterozygote superiority also could result from overestimation of effects as
pointed out by Lande and Thompson (1990) and Lande (1992). Stuber et al. (1992) and
Paterson et al. (1991) found little evidence for epistasis in maize and tomato (Lycopesicon
esculentum [E]), respectively. Paterson et al. (1991) concluded that molecular marker-
mapping studies conducted to date may identify preferentially QTL that function
independently of unlinked genetic factors. Those researchers suggested that the role of
epistasis in quantitative inheritance needs to be studied in larger populations, with more
closely spaced markers, and/or with specially constructed genetic stocks carrying particular
QTL. Stuber et al. (1992) found limited evidence for G x E interaction even though a
standard analysis of the data revealed significant G x E interaction for many of the traits.
Thus it may be possible to detect QTL with large effects in relatively few environments.
Paterson et al. (1991) reported that individual QTL showed a range of sensitivities to
environments, some QTL beingn detected in all environments and some being detected in
only one environment
.
Application of molecular markers, micro-level tests and interclass hybridizations in
improving wheat grain quality
Hard and soft wheats are two distinct classes of wheat, and generally crosses are made
within class because of distinct quality goals. For example, hard wheats with strong gluten
and high protein content are preferred for bread and chapati making and soft wheat with weak
gluten and low protein content for biscuit, cakes, pretzels and white salted noodles. This has
restricted combinations of desirable alleles from both the classes and hence reduced the
genetic base. Analysis of large numbers of wheat varieties released in India during the last
100 years showed the predominance of hard wheats with null mutation in puroindoline A.
This shows that within class (hard) hybridizations had been attempted in majority of crosses
in India in wheat breeding. Moreover, during the past two decades wheat breeders have used
the short arm of rye chromosome 1R as source of genes for disease and pest resistance and
improved agronomic performance. However, reduced gluten strength and loaf volume and
increased dough stickiness have been reported associated with 1B/1R translocation.
Therefore, combining desirable alleles using interclass hybridizations would expand genetic
bases and improve the chances of wheat improvement for yield and quality traits. This can be
accomplished by utilizing the existing knowledge of genetic basis of major grain quality traits
such as grain hardness, protein content and gluten strength at molecular level as well as their
phenotypic assessment using micro-level tests in early segregating generations.

Application of molecular markers to aquaculture and brood stock management with

special emphasis on microsatellite DNA
The use of molecular genetic markers to address questions related to aquaculture
management has steadily grown over the last two decades. These markers can provide
valuable information for various aspects of aquaculture practice, such as: (i) genetic
identification and discrimination of aquaculture stocks; (i¡) monitoring of inbreeding or other
changes in the genetic composition of the stocks that may result from such phenomena as
breeding programmes, founder events and genetic drift; (iii) comparisons between hatchery
and wild stocks; (¡v) assessment of the impact on natural populations of escaped or released
cultured fish; (v) assignment of progeny to parents through genetic tags, so that animals from
different families can be reared together in breeding programmes; (vi) identification of
marker genetic loci associated with quantitative trait loci (QTL) and use of these markers in
selection programmes (marker assisted selection); and (vii) assessment of successful
implementation of genetic manipulations such as induction of polyploidy and gynogenesis.
Allozymes were the first molecular genetic tools useidn aquaculture management.
Mitochondrial DNA (mtDNA) provided another valuable marker, because of increased
resolution and discrimination power in studies of population structure. Nuclear DNA markers
were more recently introduced in aquaculture studies and their main attractiveneslasy in their
abundance in the genome, their Mendelian inheritance, and their potential to detect high
levels of polymorphism. Especially microsatellite DNA analysis revolutionized the use of
molecular genetic markers in aquaculture and seems to be the marker destined to dominatteh
is type of studies in the coming years.

Application of molecular markers (RAPD, AFLP and Microsatellites) to Iberian pig

genotype characterization
Use of molecular markers is specially interesting for the genetic study of the Iberian
pig, whose morphologic and physiologic variability prevents the knowledge of his
populational structure. Crossbreeding, mainly with Duroc, is a common practice to increase
the carcass performance which leads to the attainment of worse quality cured products.
Molecular analysis techniques allow the estimation of genetic variability and divergence
between species and populations, and thus they can be used for phylogenetic studies,
conservation programs and control of the genetic origin of products. This work presents the
results obtained with the application of three different techniques of DNA polymorphisms
detection to samples from different populations of Iberian pigs. The RAPD technique was
used for the identification of diagnostic markers which allowed the detection of Duroc genes
in Iberian pig samples. AFLP technique was used for the attainment of specific markers of
one Iberian pig population, with a high inbreeding level, in order to study its possible
utilization in a conservation program. Finally, seven populations have been analysed with 19
microsatellite markers. The analysis of these polymorphic loci genotypes enabled the
estimation of different parameters of genetic diversity (heterozygosity, genetic distances).

MOLECULAR MARKERS AND THEIR APPLICATIONS IN CEREALS BREEDING

The development of molecular techniques for genetic analysis has led to a great
increas in our knowledge of cereal genetics and our understanding of the structure and
behaviour of cereal genomes. These molecular techniques, in particular the use of molecular
markers, have been used to monitor DNA sequence variation in and among the species and
create new sources of genetic variation by introducing new and favourable traits from
landraces and related grass species. Improvements in marker detection systems and in the
techniques used to identify markers linked to useful traits, has enabled great advances to be
made in recent years. While RFLP markers have been the basis for most work in crop plants,
valuable markers have been generated from RAPDs and AFLPs. Simple sequence repeats
(SSR) or microsatellite markers have been developed more recently for major crop plants and
this marker system is predicted to lead to even more rapid advances in both marker
development and implementation in breeding programs. Identification of markers linked to
useful traits has been based on complete linkage maps and bulked segregant analysis.
However, alternative methods, such as the construction of partial maps and combination of
pedigree and marker information, have also proved useful in identifying marker/trait
associations.

Genotype identification and genetic diversity

It is vital for plant breeding programmes to have sufficient diversity available to allow
for the production of new varieties that are aimed towards the improvement of crop
productivity and able to withstand damage from biotic and abiotic factors. In this respect,
efforts have also been made to predict the prospects of developing superior genotypes from a
cross by the measurement of genetic similarity (GS) or genetic distance (CD) between the
parents, since the later can be used as an estimation of expected genetic variances in different
sets of segregating progenies derived, from different crosses.
DNA fingerprinting of cereals species and cultivated varieties has a long scientific
history. When DNA profiling technology first came into use, restriction fragment length
polymorphism (RFLP) were considered state-of-art. RFLP technology was followed random
amplification of polymorphic DNA (RAPD), followed by amplified length polymorphism
(AFLP) and most recently use microsatellite markers or single sequence repeat (SSR).
Advantage of SSR markers are:
- the method is relatively simple and can be automated;
- most of the markers are monolocus and show Mendelian inheritance;
- SSR markers are high informative;
- a high number of public SSR primer pairs are available;
- effective cost per genotype and primer (similar to that for RAPD).

Trait tagging and marker-assisted selection

A large number of cereal studies have used markers as a tool to identify major genes,
QTLs, or to introduce new characters in elite germplasm. In wheat, for example, molecular
markers have been identified that are associated with around 40 traits of economic
importance. Knowing the location of these genes/traits and specific alleles offers the
possibility to apply marker-assisted selection (MAS) in cereals, because one of the main
objectives of plant breeding is the introgression of one or more favourable genes from a
donor parent into the background of an elite variety. Marker-assisted selection allows plant
selection at the juvenile stage from an early generation. For simply inherited traits,
conventional PCR, which requires a small amount of DNA, is becoming very useful for
screening large populations of segregating progenies. Unfavourable alleles can be eliminated
or greatly reduced during the early stages of plant development through MAS, focusing the
selection in the field on reduced numbers of mature plants.

Application of molecular markers in breeding for resistance to Barle yellow mosaic virus.
 Barley yellow mosaic virus disease – caused by Barley mild mosaic virus (BaMMV)
and Barley yellow mosaic virus (BaYMV) – has to be considered as one of the most important
diseases of winter barley in Europe and East Asia. Because of the transmission by the
soilborne fungus Polymyxa graminis, chemical treatments to control the disease are neither
efficient nor economic. Therefore, breeding for resistance to this disease is of special
importance. However, field selection for resistance genotypes is often difficult to perform
because of unpredictable environmental conditions. Consequently, the application of closely
linked PCR-based markers for the transmission of resistance gene(s) against barley yellow
mosaic virus is now successful and efficient.

Application of molecular markers in breeding for resistance to Fusarium head blight in

wheat
Fusarium head blight is a serious disease of wheat (Triticum aestivum L.) in humid
and semi humid areas of the word. In Central Europe, severe natural epidemics of Fusarium
head blight (FHB) occur once or twice in a decade and can sharply reduce yield and quality
of susceptible genotypes. Deoxynivalenol (DON) are harmful to humans, because they are
highly heat stable and cannot be eliminated totally once they entered the food chain.
Evaluation of Fusarium head blight resistance is time consuming, laborious and costly
because the inheritance of resistance is complex and phenotypic expression is significantly
affected by environmental factors. Molecular markers closely linked to the major QTL
involved in FHB resistance have recently been found and raise the possibility of using MAS
for introducing resistance alleles into elite wheat varieties as have been confirmed also by us.
However, do to the multifactorial nature of FHB resistance, the combination of MAS on the
major QTL during seedling stage with phenotypic selection on the particular plants after
flowering stage could be at the moment more sufficient and safety strategy in breeding of a
new varieties combining a high level of yield performance and high level of resistance to
Fusarium head blight.

Conclusion
In recent times many DNA markers have been developed and are powerful tools for
successful cereal breeding. The promise of marker-assisted selection in crop breeding still
remains but achieving practical benefits is taking longer than expected. The main reasons for
this delay are the insufficient quality of markers (regarding their predictive and/or diagnostic
value), inadequate experimental design, high costs and complexity of quantitative traits.
Molecular markers may complement plant breeding in three general ways. First,
molecular markers provide a reliable genetic-diversity measure that can be used for
determining relations among inbred lines and cultivars, assessing changes in genetic diversity
over time, protecting intellectual property rights, registering germplasm in countries that have
ratified rules of the UPOV convention, evaluating new germplasm for its potential to increase
genetic diversity, and selecting parents to hybridize in a breeding program. Second,
molecular markers through their linkage with alleles with large effects (qualitative traits) and
alleles with small effects (quantitative traits) may improve screens for many traits. Third,
molecular markers will provide the first understanding of the biology and the architecture of
many traits, particularly of quantitative traits.
 Adaptation and application of molecular markers to plant improvement will be unique
for each species and breeding program. Theoretically, many of the proposed applications of
molecular markers are viable. The question is whether they will improve the efficiency and
the cost effectiveness of a breeding program. This question can be answered only on a case-
by-case basis. Factors such as cost of the molecular marker technology, turnaround time in
the lab, cost of measuring a trait, etc. all will determine if and how markers are used in
breeding programs. Species in which traits are measured by processing through a commercial
factory or species with very long generation times clearly will benefit from applications of
molecular-marker technology. For species, such as annual grains and cereals, the situation is
ambiguous. One of the primary contributions of molecular markers will be an expansion of
our knowledge of genetics and of genome organization. This type of knowledge obviously
will improve our scientific understanding of many plant breeding problems, but the direct
effect on plant improvement will be intangible and difficult to measure.

Other important applications of molecular markers are as follows:

• Assessment of genetic variability and fingerprinting of genotypes
• Mapping of monogenic and qualitative trait loci (QTL) of economically important traits
• Estimation of genetic distance or degree of relatedness between population, inbreds and
breeding materials or among groups of accessions in germplasm
• Identification of sequences for candidates genes and economic breeding traits
• Molecular breeding for stress tolerance – abiotic and biotic
• Marker assisted selection for crop improvement in tissue cultured plant species
• Genetic purity testing in micropropagation - Micropropagated tree species
• Exs itu and in situ conservation of plant genetic resources (PGRs) and genetic diversity
conservation
• Characterization and evaluation of plant germplasm – self incompatibility and male sterility,
female sterility of economically important species
• Characterization and identification of genetic resources of perennial crops
• To investigate mechanisms that underlie somaclonal variation in the nuclear, mitochondrial
and chloroplast genomes
• To determine the origin of regenerants such as microspore or anther culture, protoplast
fusion
• Clonal propagation and stability – to avoid “segregation distortion” in anther culture derived
plants, inturn increasing survival of anther culture clones
• Detection of epigenetic changes in storage and conservation (i.e. detect duplications, seed
mixtures, inadvertent outcrossing and genetic drift)
• Study of “Anther culture” developed (double haploid) plants for gene mapping
• Screening transgenic plants for resistance genes using linked molecular markers
• Cloning and analysis of cDNA encoding a specific synthesis, thus understanding the nature
of basic control mechanisms
• Simulations and models on the detection of gene and

REFERENCES
Attardi, G. (1985). Animal mitochondrial DNA: An extreme example of genetic economy.
Intern. Rev. Cytol., 93:93- 145.
Avise , J.A. (1994). Molecular markers, natural history and evolution. Chapman & Hall, New
York.
Baker, C.S., MacCarthy, M., Smith, P.J., Perry, A.P. and Chambers, G.K. (1 992). DNA
fingerprints of orange roughy, Hoplostethus atlanticus: A population comparison. Mar. Biol.,
1 13: 561 -567.
Basaglia, F. (1992). Comparative examination of of soluble red muscle proteins of fifteen
Sparidae species. J. Fish Bio/., 40:557-566.
Basaglia, F. and Marchetti, M.G. (1991). Study of the soluble white muscle tissue proteins
from fifteen Sparidae specieJs.. Fish Biol., 38:763-772.
Batargias C. and Zouros E. (1993). Development and the potential applications of highly
polymorphic microsatellite and cDNA nuclear markers in gilthead seabream, Sparus aurata,
In: Fourth Congress of the European Society for the Evolutionary Biology, Evolution 93, 22-
28 August, Montpellier, France.
Barton, N. H. 1990. Pleiotropic models of quantitative variation. Genetics 124:773-782.
Barton, N. H., and M. Turelli. 1989. Evolutionary quantitative genetics: How little do we
know? Annu. Rev. Genet. 23:337-370.
Beavis, W. D., D. Grant, M. Albertsen, and R. Fincher. 1991. Quantitative trait loci for plant
height in four maize populations and their associations with qualitative genetic loci. Theor.
Appl. Genet. 83:141-145.
Beckman, J. S., and M. Soller. 1986. Restriction fragment length polymorphisms and genetic
improvement of agricultural species. Euphytica 35:111-124.
Batargias, C., Kotoulas, G., Magoulas, A. and Zouros E. (1997). Retorspective parentage
identification in a mass spawning of gilthead sea bream, by means of microsatellite DNA.
Preliminary results of genetic selection. In: Fifth Panhellenic Congress of Oceanography and
Fisheries, Kavala, Greece, 15-18 April 1997 (in greek).
Blouin, S. M., Parsons, M., Lacaille, V and Lotz, S. (1996). Use of microsatellite loci to
classify individuals by relatednessM. ol. €col., 5: 393-401.
Brown, D.M., Prager E.M., Wang, A., and Wilson, A.C. (1982). Mitochondrial DNA
sequences of primates: tempo and mode of evolution. J. Mol. €vol., 18:225-239.
Carvalho, G.R. and Hauser, L. (1994) Molecular genetics and the stock concept in fisheries.
Rev. Fish. Biol. Fish., 4:326-350.
Cox, T. S. 1992. Use of molecular markers for analyzing quantitative traits in wheat. In: Proc.
19th Hard Red Winter Wheat Workers Conf.
Kinoshita, T. 1990. In: Report of the committee on gene symbolization, nomenclature and
linkage groups. RGN 7: 16-57.
Lande, R. 1992. Marker-assisted selection in relation to traditional methods of plant breeding.
p.437-451. In: H. R. Stalker and J. P. Murphy (eds.) Plant breeding in the 1990s. CAB
International, Wallingford, UK.
Lande, R. and R. Thompson. 1990. Efficiency of marker-assisted selection in the
improvement of quantitative traits. Genetics 124:743-756.
Stuber, C. W., S. E. Lincoln, D. W. Wolff, T. Helentjaris, and E. S. Lander. 1992.
Identification of genetic factors contributing to heterosis in a hybrid from two elite maize
inbreds using molecular markers. Genetics 132:823-839.
Weising, K., Nybom, H., Wolff, K. and Meyer, W. 1995. In: DNA Fingerprinting in Plants
and Fungi (ed. Arbor, A.). CRC Press, Boca Raton.
von Wettstein-Knowles, P. 1992. Cloned and mapped genes: Current status. pp. 73-98. In P.
R. Shewry, (ed.), Barley: In: Genetics, Biochemistry, Molecular Biology and Biotechnology.
Alden Press, Oxford.
Melchinger, A. E. 1990. Use of molecular markers in breeding for oligogenic disease
resistance.Plant Breeding 104:1-19.
Melchinger, A.E. 1993. Use of RFLP markers for analyses of genetic relationships among
breeding materials and prediction of hybrid performance. Proc. of the First Int. Crop Sci.
Cong.
Buerstmayer, H., Lemmens, M., Hartl, L., Doldi, L., Steiner, B., Stierschneider, M. and
Ruckenbauer P. Molecular mapping of QTLs for Fusarium head blight resistance in spring
wheat. I. Resistance to fungal spread (Type II resistance). Theor. Appl. Genet. 104 (2002) 84-
91.
Gurta, P.K., Varshney, R.K., Sharma, P.C. and Ramesh, B. Molecular markers and their
applications in wheat breeding. Plant Breed 118 (1999) 369-390.
Korzun, V., Malyshev, S., Voylokov, A.V. and Börner, A A genetic map of rye (Secale
cereale L.) combining RFLP, isozyme, microsatellite and gene loci. Theor. Appl. Genet. 102
(2001) 709-717.
Röder, M.S., Korzun, V., Wendehake, K., Plaschke, J., Tixier, M., Leroy, P. and Ganal,
M.W. A microsatellite map of wheat. Genetics 14 9 (1998) 2007-2023
Paterson, A. H., S. D. Tanksley, and M. E. Sorrells. 1991. DNA markers in plant
improvement. Adv. Agron. 46:39-90.
Pollak, E., O. Kempthorne, and T. B. Bailey, Jr. 1977. Proceedings of the first international
conference on quantitative genetics. Iowa State University Press, Ames.
Robertson, D. S. 1985. A possible technique for isolating genic DNA for quantitative traits in
plants. J. Theor. Biol. 117:1-10.